1. General
This specification contains nucleotide and amino acid sequence information prepared using PatentIn Version 3.3, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term “SEQ ID NO:”, followed by the sequence identifier (e.g. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
Each embodiment described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference:                1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;        2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;        3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;        4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; and        5. Perbal, B., A Practical Guide to Molecular Cloning (1984).        
2. Description of the Related Art
Cancer is a major cause of morbidity throughout the world. For example, in 2001, the American Cancer Society estimated that 553,768 Americans died from a form of cancer. Cancer is responsible for 22.9 percent of all American deaths and is exceeded only by heart disease as a cause of mortality.
All studied forms of cancer share the characteristics abnormal cell division, growth, and differentiation. The initial clinical manifestations of cancers are generally heterogeneous, with over 70 types of cancer arising in each of a number of organs and tissues of the human body. Moreover, while some cancers may appear clinically similar they may actually represent different molecular diseases. This diversity in clinical and molecular characteristics make cancer difficult to diagnose. As a consequence, a variety of assays are required to detect even a small number of the known cancers.
Family history still remains the most reliable diagnostic procedure for identifying patients at risk of cancer.
Cancer surveillance has been effective for detecting some cancers in which risk can be identified, for example colorectal cancer in familial adenomatous polyposis coli and hereditary nonpolyposis colorectal cancer (Markey et al., Curr. Gastroenterol. Rep. 4: 404-413, 2002), but these syndromes cumulatively account for less than 1% of cancer patients (Samowitz et al., Gastroenterology 121: 830-838, 2001). Nevertheless, genetics is thought to contribute substantially to cancer risk, since the odds ratio for malignancy increases in patients with first degree relatives with cancer, e.g., 2 to 3-fold in colorectal cancer (Fuchs et al., N. Engl. J. Med. 331: 1669-1674, 1994). Therefore, there remains a need to develop genetic tests to identify these patients.
The detection of microsatellite instability as a diagnostic for cancer, requires the patient to have a detectable tumor beforehand and, as a consequence, is not an early test that can lead to early effective treatment. Microsatellite instability compares microsatellite marker length between the monoclonal tumor cell population and normal tissue derived from the same patient. As microsatellites are unstable in the population, an assay measuring such instability must include a control sample from the same subject. This leads to increased cost, as a number of samples must be assayed for each diagnosis performed.
Genetic changes that are associated with cancer include gene mutation in critical tumor-associated genes, as well as gene deletion or loss of heterozygosity (LOH) of larger regions harboring tumor suppressor genes. In addition to genetic changes it is clear that epigenetic changes are also a common hallmark of cancer DNA, with changes in both DNA methylations and histone modification of the CpG island regions spanning the promoters of tumor suppressor genes (Jones and Baylin, Nat. Rev. Genet. 3: 415-428, 2002). However, it is not clear as to the extent and nature of these epigenetic changes in cancer cells.
Changes in the state of methylation of DNA have been observed in cancer cells (Feinberg et al., Nature, 301: 89-92, 1983), including the loss of methylation at normally methylated sequences (hypomethylation) and the gain of methylated sequences at sites that are usually non-methylated (hypermethylation). For example, global hypomethylation has been reported in almost every human malignancy studied to date (Feinberg et al., supra and Bedford et al., Cancer Res., 47: 5274-5276, 1987). More particularly, Gama-Sosa et al., (Nucl. Acids Res., 11: 6883-6894) measured the levels 5-methylcytosine content by HPLC and showed a reduced level in cancer tissues compared to control tissues. However, the 5-methylcytosine content of a cell is not necessarily a measure of the level or extent of chromatin modification in these cells. Accordingly, the assay of Gama-Sosa et al., does not provide an accurate measurement of chromatin changes that are associated with cancer.
The major site for methylation in mammals is a cytosine located next to a guanine (5′-CpG-3′), including a so-called “CpG island”. Generally, these targets of methylation are not distributed equally in the genome, but found in long GC-rich sequences present in satellite repeat sequences, middle repetitive rDNA sequences and centromeric repeat sequences. CpG islands are generally recognised as sequences of nucleic acid that comprise a GC content of over 50% (in contrast to a genome-wide average in humans of about 40%) and an observed over expected ration of CpG of 0.6 or greater (Gardiner-Garden and Frommer, J. Mol. Biol., 196: 261 to 281, 1987; Takai and Jones, Proc. Natl. Acad. Sci. USA, 99: 3740-3745, 2002).
CpG islands can become de novo methylated in a cancer cell and this is associated with gene silencing. DNA hypermethylation of the CpG island region is also accompanied by local changes in histone modification, including de-acetylation and methylation of the lysine 9 residue of Histone H3 (K9-H3).
For example, CpG islands within the promoter regions of specific genes can be hypermethylated in some cancers, e.g., in the case of BRCA1 promoter hypermethylation in breast cancer (Dobrovic et al., Cancer Res., 57: 3347-3350, 1997) and the VHL gene promoter hypermethylation in clear cell renal carcinomas (Herman et al., Proc. Natl. Acad. Sci. USA, 91: 9700-9704). However, the methylation of these genes is limited to discrete regions of these genes and shown to be useful only in relation to the detection of specific cancers (Plass, Hum. Mol. Genet., 11: 2479-2488, 2002).
It is widely recognized that simple and rapid tests for the early detection of cancers, especially multiple cancer types, have considerable clinical potential. In view of the heterogeneity of cancers, it is difficult to produce a single diagnostic that is useful for different cancer types. Such tests have potential use for an initial diagnosis, as well as for determining prognostic outcomes e.g., for detecting tumor recurrence following surgical resection and/or chemotherapy. A molecular diagnostic approach that identifies patients with cancer or at risk of cancer, would offer a decisive advantage for intervention and treatment.